Vascular endothelium constitutively generates nitric oxide (NO) in large vessels and induces a relaxation of smooth muscle cells. However, little is known about the production of NO in microvessels, where smooth muscle layers are thin or absent. In this study, we have compared the constitutive production of NO in bovine brain microvascular endothelial cells (BBEC) with that in bovine aortic endothelial cells (BAEC). ATP, acetylcholine (ACh) and A23187 induced Ca²⁺ transients both in BBEC and BAEC. In contrast, ATP and A23187 that evoked similar degree of [Ca²⁺] _i increase failed to induce NO production in BBEC, as measured with an NO-sensitive fluorescent dye DAF-2, whereas BAEC showed an increase in DAF-2 fluorescence. Hypotonic stress induced ATP release and subsequent NO production in BAEC, whereas these were not observed in BBEC. Furthermore, we have developed an in vitro model vessel system that consists of aortic smooth muscle cells-embedded collagen gel lattice and overlaid endothelial cells. Precontracted gels showed relaxation in response to ACh, when BAEC were overlaid. However, ACh-induced relaxation was not observed in BBEC-overlaid gels. Expression of eNOS protein as well as cellular uptake of L-[³H] arginine were significantly lower in BBEC than in BAEC. These results indicate that Ca²⁺-dependent NO production is an undetectable level in BBEC, for which at least two factors, i.e., low levels of eNOS expression and L-arginine uptake, are responsible.